As a typical technique for immobilizing a physiologically active substance on an measurement chip, a method (amine coupling method) that involves binding an amino group of a physiologically active substance to a carboxyl group on a measurement chip is broadly used. This method requires dissolving a physiologically active substance in a buffer having a pH that is lower than the isoelectric point of such substance upon immobilization. Specifically, whereas a physiologically active substance will be positively charged when the pH is the isoelectric point of such substance or lower, a carboxyl group on a measurement chip is negatively charged from the alkali side through the acidic region with approximately pH 3.5. Therefore, a physiologically active substance is concentrated on a measurement chip due to electrostatic attraction. When such preconcentration is not performed, the amount of a physiologically active substance immobilized will drastically decrease. Thus, a physiologically active substance to be immobilized should be dissolved in a buffer having a pH that is lower than the isoelectric point of such substance, as disclosed in J. C. S. Chem. Commun., 1990, 1526 and U.S. Pat. No. 5,436,161.
This means that a physiologically active substance that is denatured under low-pH conditions is unable to be immobilized while maintaining its activity. Furthermore, a physiologically active substance such as an acidic protein has no positive net charge, even in the case of a pH of approximately 3.5. Thus, no preconcentration effects can be obtained, so that immobilization becomes impossible.
A physiologically active substance dissolved in a buffer having a pH that is higher than the isoelectric point of such substance can be immobilized on a solid surface because of electrostatic attraction between the substance and a cationic polymer immobilized on the solid surface. JP Patent Publication (Kokai) No. 8-245815 A (1996) discloses a technique using such principle, which involves alternately layering a protein and an organic polymer ion.
This method is very good in that a physiologically active substance can be conveniently immobilized. However, two problems arise in view of application to a biosensor. The first problem is that because binding between a protein and a substrate depends only on electrostatic interaction, a part of the physiologically active substances that have been electrostatically adsorbed on a solid surface may be dissociated due to a washing step using an acidic solution or an alkaline solution. The second problem is that a physiologically active substance is obtained in the form of a densely packed monomolecular layer. To increase the amount of a physiologically active substance immobilized, it is desirable to three-dimensionally immobilize a physiologically active substance. Furthermore, dense packing of a physiologically active substance is not preferable in terms of application to a biosensor for assaying binding and dissociation behaviors of compounds interacting with the physiologically active substance.